The present invention relates to electrochemical sensors comprising at least one working electrode, a counter electrode and a reference electrode, for which one works at a constant potential, and in which one measures the current circulating between the working electrode and the counter electrode, caused by an oxidation reduction reaction at the working electrode. As a result, the invention does not relate to sensors based on potentiometric measurements, of the Ion-Selective Electrode type, comprising two electrodes, i.e. a working electrode and a reference electrode, between which a difference in potential is measured. Such sensors are described in the publication by UI Hague et al. A MEMS fabricated cell electrophysiology biochip for in silico calcium measurements, Sensors and Actuators B; Elsevier Sequoia S A. Lausanne, C H, vol. 123, no. 1, 2007 Mar. 30, pages 391-399, in the publication by Neuman M R et al. Batch-produced microfabricated ion-selective electrodes, Engineering in medicine and biology society, 1995, IEEE 17th Annual Conference Montreal, Vol. 2, 1995 Sep. 20, pages 1557-1558, and in the publication by Wang S-H et al, Development of a solid-state thick film calcium ion-selective electrode, Sensor and Actuators B, Elsevier Sequoia S A; Lausanne C H, vol. 96, no. 3, 2003 Dec. 1, pages 709-716. These sensors are used in the field of biomedical measurements, to measure the pH, the potassium or calcium concentration in biological fluids.
The invention also does not relate to sensors for which one measures a current, but by varying the potential over time, such as sensors using the Square Wave Anodic Stripping Voltammetry (SWASAV) technique to measure the heavy metals, described in the publication by KIM H-J et al. A direct analysis of nanomolar metal ions in environmental water samples with Nafion-coated microelectrodes, Electrochimica Acta, Elsevier Science Publisher, Barking G B, Vol. 50, no. 1, 2004 Nov. 15, pages 205-210. Such sensors containing mercury are not in compliance with environmental standards.
Amperometric sensors of the type of that of the present invention are for example described in patent EP 0 586 982. Patent EP 0 586 982 describes a first type of integrated sensor comprising an insulating substrate on which three electrodes are formed, i.e. a working electrode, a counter electrode and a reference electrode. The electrodes are then covered with a diffusion membrane, which covers all of the three electrodes.
Another type of sensor is also described and comprises an insulating substrate comprising an insulating layer, in which openings are formed. Each opening receives a metal deposition intended to form one of the electrodes. A diffusion membrane completely covers the active conducting part of the working electrode by overhanging through its entire peripheral area.
One such type of sensor requires adapting the insulating layer to each alternative embodiment of the electrodes, and therefore modifying the entire manufacturing method as a function of the desired shape of the electrodes.
Furthermore, the arrangement of the layers requires providing for a contact zone under the electrodes. Moreover, the geometric dimension of the working electrode must be sufficient, from several hundred micrometers to one millimeter, to make it possible to deposit the membrane there.
Moreover, sensors are known that are used to measure protein or glucose concentrations. These species not being electroactive, it is necessary to use a membrane in which an electroactive species is immobilized serving as intermediate species for the reaction. This additional species, used for the measurement at the working electrode, is therefore not dissolved in the medium. Such sensors are described in the publication by Kim P et al. An electrochemical interface for integrated biosensors. IEEE International Conference on sensors, New York, vol. CONF. 2, 2003 Oct. 22, pages 1036-1040 vol. 2, and in the publication by Alonso Lomillo M A et al Biosensor Based on Platinum Chips for Glucose Determination, Analytica Chimica Acta, Elsevier, Amsterdam, N L, vol. 547, no. 2, 2005 Aug. 22, pages 209-214. Unlike the amperometric sensors of the inventive type for which the reaction occurs at the working electrode, these sensors comprise a membrane that constitutes the reaction site for the undissolved electroreactive species, the sensor serving to detect the current of that reaction. Such sensors have the drawback of having a limited lifetime, as the electroreactive membrane becomes charged with species to be measured and tends to leak after a certain usage time.
One aim of the present invention is therefore to offset these drawbacks, by proposing an amperometric sensor that can be made using a simple manufacturing method and whereof only one step must be modified to manufacture electrodes having the desired shape, all of the other steps of the method being the same irrespective of the type of electrodes.
Another aim of the present invention is to propose a sensor whereof the lifetime is improved, in particular by increasing the adhesion of the polymer membrane used as diffusion membrane on a selected insulator.
Another aim of the present invention is to propose a manufacturing method making it possible to produce a nanometric insulating structure between the membrane and the electrodes.
Another aim of the present invention is to propose a sensor making it possible to directly access the electrodes to make the connections.
Another aim of the present invention is to propose a sensor making it possible to have a substrate other than a silicon substrate, and in particular a transparent substrate.
Another aim of the present invention is to propose a sensor making it possible to be miniaturized, without liquid electrolyte, and to use two same sensors in a same probe in a limited space.